AIRWAY BIOLOGY Dendritic cell subsets in human ... · Human dendritic cells are identified by the abundant expression of major histocompatibility complex class II (HLA-DR) and the

AIRWAY BIOLOGY

Dendritic cell subsets in human bronchoalveolar lavage fluidafter segmental allergen challengeKai Bratke, Marek Lommatzsch, Peter Julius, Michael Kuepper, Hans-Dieter Kleine, WernerLuttmann, J Christian Virchow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figure A and table A areavailable at http://thorax.bmjjournals.com/supplemental

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr M Lommatzsch, Abteilungfur Pneumologie, Klinik undPoliklinik fur Innere Medizin,Universitat Rostock, Ernst-Heydemann-Str. 6, Rostock18057, Germany;[email protected]

Received 26 June 2006Accepted 4 August 2006Published Online First23 August 2006. . . . . . . . . . . . . . . . . . . . . . . .

Thorax 2007;62:168–175. doi: 10.1136/thx.2006.067793

Background: Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in humanbronchoalveolar lavage fluid (BALF) after allergen challenge are unknown.Methods: 7 patients with allergic asthma (median 23 years, range 19–25 years) underwent segmentalchallenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers inBALF and in peripheral blood were analysed using four-colour flow cytometry.Results: Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01–0.08%) and myeloid dendritic cells(mDCs, median 0.47%, range 0.27–0.87%) were detectable in BALF from control segments. CD1a-positivedendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09–1.83%) and mDCs (median 1.82%, range 0.95–2.29%) increased significantly in BALF 24 h (p = 0.018compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. Thepercentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by anenhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease inmDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis ofdendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less maturephenotype compared with BALF dendritic cells from control segments.Conclusion: Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shownfor the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen afterallergen challenge in patients with asthma.

Dendritic cells form a highly sensitive sentinel network inthe lung. By continuously reporting antigenic informa-tion from the airways to pulmonary lymph nodes, they

are capable of upregulating or downregulating specific immuneresponses in the lung.1 2 Animal models suggest that dendriticcells have a crucial role in asthma.3 Allergen challenge results ina recruitment of dendritic cells into the airways, which isessential for the establishment of allergic airway inflamma-tion.4 5 Instillation of allergen-loaded dendritic cells into theairways alone can induce allergic airway inflammation inhealthy animals.6 Selective elimination of dendritic cells duringallergic airway inflammation abolishes the characteristicfeatures of asthma, including eosinophilic inflammation, gobletcell hyperplasia and bronchial hyperresponsiveness.7 These dataindicate that dendritic cells are vital for both the initiation andthe maintenance of allergic airway inflammation in asthma.8

Human dendritic cells are identified by the abundantexpression of major histocompatibility complex class II (HLA-DR) and the absence of lymphocyte, natural killer cell,monocyte and granulocyte lineage markers.2 CD11c+ myeloiddendritic cells (mDCs) and CD123+ plasmacytoid dendritic cells(pDCs) represent two main dendritic cell subtypes. There isongoing research to further characterise these two dendritic cellsubtypes in human lung parenchyma9–11 and in humanbronchoalveolar lavage fluid (BALF).12 13 Patients with asthmadisplay increased numbers of intraepithelial dendritic cells inthe airways compared with healthy individuals.14 15 Allergenchallenge causes an accumulation of CD11c+ mDCs within thebronchial mucosa,16 and a parallel decline of these cells in

circulating blood.17 In contrast, CD123+ pDC were postulated tobe absent from the bronchial mucosa, both before and afterallergen challenge,16 and have not yet been studied inperipheral blood after allergen challenge.17 However, localincreases in pDCs have been reported in patients with atopicrhinitis and dermatitis after allergen challenge,18 19 as well aselevated pDC counts in the peripheral blood of patients withallergic asthma.20 The relative contribution of mDCs and pDCsto the pathology of human asthma is, therefore, still unre-solved. In addition, no information is available on theaccumulation and the characteristics of pDCs and mDCs inBALF fluid after allergen challenge in human asthma. Thisstudy investigated dendritic cell subsets in human BALF aftersegmental allergen challenge using a comprehensive flowcytometric method.

METHODSParticipantsSeven patients with mild allergic asthma (median 23 years,range 19–25 years), most of them being medical students at theUniversity of Rostock, volunteered for the study (table 1).Patients were recruited for the study on the basis of previouslydescribed criteria: (1) airway hyperresponsiveness; (2) positiveallergen skin prick tests; (3) elevated total or specific IgEconcentrations; and (4) a dual reaction after allergen

Abbreviations: BALF, bronchoalveolar lavage fluid; BDCA, blooddendritic cell antigen; mDC, myeloid dendritic cell; pDC, plasmacytoiddendritic cell

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inhalation.21 The individual provocation dose was calculated asdescribed.22 Inhaled and segmental allergen challenges wereseparated by at least 4 weeks. Corticosteroids were withdrawnat least 7 days before challenge. Patients gave their writteninformed consent. The study was approved by the local ethicscommittee.

Segmental allergen challengeSegmental allergen challenge was performed as describedpreviously.21 Briefly, 2.5 ml of saline were instilled into the leftS8 (control 1, C1) and S5 (control 2, C2) segments, and allergen(diluted in 2.5 ml of saline) was instilled into the right S8(allergen 1, A1) and S5 (allergen 2, A2) segments. The left andright S8 (C1 and A1) segments were lavaged using 100 ml ofpre-warmed saline after 10 min, and the left and right S5segments (C2 and A2) after 24 h. Median BALF recoveries wereas follows: C1 57 ml (range 40–68 ml), A1 47 ml (40–59 ml),C2 62 ml (50–75 ml) and A2 60 ml (22–64 ml). Before eachbronchoscopy, venous blood samples were obtained.

Analysis of cell counts and leucocyte subsets in BALFBALF samples were filtered through a two-layer sterile gauzeinto sterile plastic vials, centrifuged at 4 C and 500 g for 10 min.

Cells were resuspended in phosphate-buffered saline. A fractionof the suspension was used for cell counts (using a Neubauerchamber; Brand, Wertheim, Germany) and for cytospins.Cytospins were stained with May/Grunwald/Giemsa solution(Merck, Darmstadt, Germany) and differential cell countsdetermined using standard morphological criteria. Results wereexpressed as the total number of cells per ml of recovered fluid.

Flow cytometryFreshly collected EDTA blood and isolated BALF cells wereincubated with the respective antibodies (table 2) for 20 min.Afterwards, FACS lysing solution (BD Biosciences, Heidelberg,Germany) was added for another 10 min. Cells were centrifugedfor 5 min (400 g) and washed with phosphate-buffered salinecontaining 2% fetal calf serum and 0.1% NaN3. Finally, cells wereresuspended in phosphate-buffered saline and analysed on aFACS Calibur using Cell Quest Pro Software (BD Biosciences). Toidentify dendritic cell subsets in BALF, we used an approachdescribed previously to detect mDCs and pDCs in peripheralblood.20 CD3, CD14, CD16, CD19, CD20 and CD56 negative/dimcells (linneg/dim) were gated using a commercial lineage cocktailwith antibodies against all of these antigens (fig 1A). Amongthese linneg/dim cells, distinct populations of CD123+HLA-DR+ cells

Table 1 Patient characteristics

No Sex AgeFEV1

(% predicted) DrugsTotal IgE(kU/l)

Specific IgE(kU/l) Allergen Dose (AU)

1* M 23 77 BA 548 26.1 Birch 762 F 23 92 BA 564 77.1 DP 2503 F 25 94 BA, IC 96 4.0 Birch 304 F 25 105 CR, BA 64 16.9 DP 185 F 23 105 BA 545 12.6 Rye 766 F 24 102 BA 155 5.2 Rye 47 M 19 114 BA 141 18.7 Rye 600

BA, inhaled b2 agonist; CR, cromoglycate; DP, dermatophagoides pteronyssinus; F, female; FEV1, forced expiratory volume in the first second; IC, inhaledcorticosteroid; IgE, immunoglobulin E; M, male.Table 1 displays the sex (male/female) and age (in years) of the patients, the prebronchodilator forced expiratory volume in the first second (FEV1 in % predicted), thedrugs taken before the study, serum levels of total (normal range ,100 kU/l) and allergen-specific (normal range ,0.7 kU/l) immunoglobulin E (IgE) in kilo units (kU/l),and the allergen and the dose (in allergen units, AU) used for segmental allergen challenge. *Dendritic cell counts only from saline and allergen-challenged segmentsafter 24 h.

Table 2 Antibodies used for four-colour flow cytometry

Antigen Label Clone Company

Lineage cocktail CD3 FITC SK7 BD BiosciencesCD14 FITC MwP9 BD BiosciencesCD16 FITC 3G8 BD BiosciencesCD19 FITC SJ25C1 BD BiosciencesCD20 FITC L27 BD BiosciencesCD56 FITC NCAM16.2 BD Biosciences

Other antibodies CD11c PE S-HCL-3 BD BiosciencesCD11c APC S-HCL-3 BD BiosciencesCD123 PE 9F5 BD BiosciencesHLA-DR PerCP L243 BD BiosciencesCD40 APC HB14 Caltag/InvitrogenCD83 APC HB15e Caltag/InvitrogenCD86 APC BU63 Caltag/InvitrogenCD80 APC MEM-233 ImmunotoolsCD1a PE NA1/34 DakoBDCA-1 APC AD5-8E7 Miltenyi BiotecBDCA-2 APC AC144 Miltenyi BiotecBDCA-3 APC AD5-14H12 Miltenyi BiotecBDCA-4 APC AD5-17F6 Miltenyi Biotec

APC, allophycocyanin; BDCA, blood dendritic cell antigen; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP,peridinin chlorophyll protein.Caltag/Invitrogen, Karlsruhe, Germany; Dako, Glastrup, Denmark; Miltenyi Biotec, Bergisch Gladbach, Germany;Immunotods, Friesoythe, Germany.

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(pDC) and CD11c+HLA-DR+ cells (mDC) were identified, both inperipheral blood and in BALF (fig 1A). A population ofCD1a+HLA-DR+linneg/dim cells identified in BALF could hardly bedetected in peripheral blood (fig 1A). These CD1a+ dendritic cellscoexpressed CD11c in all BALF samples, suggesting that thesedendritic cells are a subpopulation of mDCs (fig 1B). Backgatingshowed that pDCs, mDCs and CD1a+ dendritic cells from BALFform distinct populations in the forward scatter/side scatter plot,without an overlap with lymphocyte, granulocyte and alveolarmacrophage regions (fig 1C).

Statistical analysisData were analysed using SPSS v 11.0. Most parameters werenon-normally distributed. Therefore, correlation analysis was

performed using Spearman’s correlation coefficient, and thecomparison of BALF parameters between allergen-challengedand saline-challenged control segments, and the comparison ofblood parameters before and after allergen challenge wasperformed using Wilcoxon’s signed rank test. The comparisonof dendritic cells in peripheral blood and BALF was performedusing the Mann–Whitney U test. Probability values of p,0.05were regarded as significant.

RESULTSDendritic cell subsets in BALF and peripheral blood aftersegmental allergen challengeIn the BALF collected 24 h after saline challenge, low amountsof pDCs (median 0.06% or 0.066103 cells/ml BALF; range

Figure 1 Identification of dendritic cell subsets in bronchoalveolar lavage fluid (BALF). (A) Total cells were identified in forward scatter/side scatter (FSC/SSC) plots (first row). Lineage negative/dim (linneg/dim) cells were further gated (second row) to identify dendritic cells. Among linneg/dim cells, plasmacytoiddendritic cells were identified by CD123 and HLA-DR expression (third row), myeloid dendritic cells by CD11c and HLA-DR coexpression (fourth row) andCD1a+ dendritic cells by coexpression of CD1a and HLA-DR (fifth row). (B) BALF-derived CD1a+ dendritic cells were gated and analysed for CD11cexpression (black) compared with an isotype control antibody (grey). (C) Backgating of dendritic cell subsets showed distinct cell populations (blue) in theFSC/SSC plot.

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0.01–0.08% or 0.01–0.346103 cells/ml BALF), mDCs (median0.47% or 0.636103 cells/ml BALF; range 0.27–0.87% or 0.25–3.26103 cells/ml BALF) and CD1a+ mDCs (median 0.34% or0.566103 cells/ml BALF; range 0.24–0.56% or 0.25–1.636103 cells/ml BALF) were detected. There were no sig-nificant differences in dendritic cell counts in BALF 10 minafter saline challenge (fig 2). In all patients, the percentages aswell as the total numbers of pDCs (median 0.56% or2.716103 cells/ml BALF; range 0.09–1.83% or 0.06–16.296103 cells/ml BALF; p = 0.018 compared with the corre-sponding control), mDCs (median 1.82% or 7.456103 cells/mlBALF; range 0.95–2.29% or 0.60–20.386103 cells/ml BALF;p = 0.018) and CD1a+ mDCs (median 1.44% or 5.226103 cells/ml BALF; range 0.63–2.00% or 0.40–17.806103 cells/ml BALF;p = 0.018) increased significantly in BALF 24 h, but not10 min, after allergen challenge (fig 2). The percentage increasein pDCs was higher than the percentage increase in mDCs 24 hafter allergen challenge in all patients. This was reflected by a

significantly enhanced pDC:mDC ratio in allergen-challengedsegments (median 0.36, range 0.09–0.80) as compared withcontrol segments (median 0.11, range 0.04–0.23) after 24 h(p = 0.018). In peripheral blood, distinct populations of pDCsand mDCs were detected before (median of pDCs 0.31%, range0.18–0.36%; median of mDCs 0.26%, range 0.17–0.36%) and24 h after allergen challenge (median of pDCs 0.14%, range0.09–0.25%; median of mDCs 0.16%, range 0.06–0.27%). Totalnumbers of pDCs decreased in four of six patients and mDCs infive of six patients in peripheral blood 24 h after challenge(fig 2). This decrease in dendritic cell counts in peripheral bloodwas statistically significant in the case of mDCs (p = 0.038), butnot in the case of pDCs (p = 0.068).

Association of dendrit ic cells with the inflammatoryresponse and allergen doseCompared with the control segments, there were significantlyincreased neutrophil (p = 0.043) and eosinophil counts

Figure 2 Dendritic cell subsets in bronchoalveolar lavage fluid (BALF) and peripheral blood after allergen challenge. Shown are percentages of dendriticcells in BALF and total dendritic cell counts in BALF and peripheral blood. A1, BALF 10 min after allergen challenge; A2, BALF 24 h after allergen challenge;B1, peripheral blood before SAP; B2, peripheral blood 24 h after SAP; C1, BALF 10 min after saline challenge; C2, BALF 24 h after saline challenge.*Significant differences between allergen challenged and corresponding control segments, and between peripheral blood before and 24 h after allergen-challenge. Median values are displayed as bars for A2, B1 and B2. mDC, myeloid dendritic cells; pDC, plasmacytoid dendritic cells.

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(p = 0.028) in BALF 24 h, but not 10 min, after challenge (seesupplimentary table A online at http://www.thorax.bmjjour-nals.com/supplemental). The amount of infiltrating dendriticcells was correlated with the severity of the local inflammatory

response, as reflected by a significant correlation between thetotal numbers of mDCs, CD1a+ mDCs and pDCs with eosinophilcounts (r.0.9 and p,0.001 for all dendritic cell subsets) andlymphocyte counts (r = 0.82–0.96, p,0.05 for all dendritic cell

Figure 3 Surface molecules on dendritic cells in bronchoalveolar lavage fluid (BALF) and peripheral blood. Plasmacytoid dendritic cells (CD123+HLA-DR+linneg/dim) and myeloid dendritic cells (CD11c+HLA-DR+linneg/dim) were gated as described in fig 1. Histograms display the expression of surfacemolecules (red) compared with corresponding isotype controls (grey). APC, allophycocyanin; BDCA, blood dendritic cell antigen.

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subsets) in BALF 24 h after challenge. There was also acorrelation between the total numbers of mDCs, CD1a+ mDCsand pDCs and the allergen dose used for challenge (r.0.86 andp,0.01 for all dendritic cell subsets).

Surface molecules on dendritic cells in BALF andperipheral bloodBoth in peripheral blood and in BALF, the expression of blooddendritic cell antigen (BDCA)-2 was restricted to pDCs,whereas BDCA-1 expression was restricted to a subset ofmDCs (fig 3, table 3). In contrast with peripheral blood, BDCA-3was expressed not only on a subset of mDCs but also on pDCs andmost BDCA-1+ mDCs in BALF. BDCA-4, which was restricted topDCs in peripheral blood, was also expressed on a subset of BALFmDCs (fig 3, table 3). This subset of BDCA-4+ BALF mDCsincreased twofold 24 h after allergen challenge compared with thecontrol segments (table 3). Of note, BDCA-3 and BDCA-4 werestrongly expressed by other BALF cells that were linbright andHLA-DR+. As eosinophils, neutrophils and macrophages wereexcluded by gating, other lineage-positive cells must account forthis expression. In addition, some expression of BDCA-3 andBDCA-4 was found on alveolar macrophages and granulocytes(see supplementary fig A online at http://www.thorax.bmjjour-nals.com/supplement). Compared with peripheral blood mDCs,the expression of CD40, CD80 and CD86 was significantly strongeron mDCs from BALF of control segments (p,0.05 in all cases;fig 3, table 3). The dendritic cell maturation marker CD83 wasabsent on blood mDCs, but detectable on approximately 25% ofBALF mDCs from the control segments. The mDCs in BALF 24 hafter allergen challenge were characterised by a significantly lowerexpression of CD83 compared with the corresponding controlsegments (p = 0.046; fig 3, table 3). Owing to the very low numberof pDCs in BALF, it was not possible to reliably analyse surfacemarkers on pDCs in the control segments. Only CD40, but notCD80, CD83 or CD86, was expressed on pDCs in peripheral blood.In contrast, a low expression of CD80 and CD86 was found onBALF pDCs 24 h after challenge. There was a non-significanttrend to a decreased expression of CD40 on BALF pDCs comparedwith blood pDCs 24 h after challenge (p = 0.09; fig 3, table 3).

Table 3 displays the percentage of marker-positive mDCs andpDCs in blood (B1, B2) or BALF (C1, C2, A2). The median

values (range) of six patients with asthma are presented. Thetime point 10 min after allergen challenge (A1) is notdisplayed, because cell counts in BALF were too low to measuresurface molecules on dendritic cells. In BALF from saline-challenged control segments (C1 and C2), numbers of pDCswere too low to quantify the expression of surface molecules.

DISCUSSIONIn this study, we have reported a comprehensive strategy forthe identification, quantification and characterisation ofplasmacytoid dendritic cells (CD123+HLA-DR+linneg/dim) andmyeloid dendritic cells (CD11c+HLA-DR+linneg/dim) in humanBALF using four-colour flow cytometry. In addition, this is thefirst study in humans to show that allergen challenge isassociated with a marked influx of both pDCs and mDCs intothe airway lumen of patients with allergic asthma. Further, itprovides a detailed analysis of the distribution of dendritic cellantigens on these infiltrating dendritic cells. Although ourstudy included a comparably small number of patients, theuniformity of the data suggests that our results are representa-tive for a larger population and that a larger number of patientswould not have changed the findings considerably.

Dendritic cells in BALF were initially identified usingimmunocytochemistry, and were postulated to yield approxi-mately 0.4% of all cells in the BALF of healthy patients.23 Insubsequent studies, the same group identified dendritic cells bytheir low autofluorescent properties in flow cytometric ana-lyses, and divided these low autofluorescent cells intosubgroups of CD1a+ and CD1a2 cells.24 25 Using flow cytometrywith a different gating strategy compared with our study,Donnenberg and Donnenberg recently described CD123+

plasmacytoid dendritic cells (0.02%) and CD11c+ myeloiddendritic cells (0.06%) in BALF from healthy volunteers.12 Ourstudy is the first to use all three markers (CD1a, CD11c andCD123) to characterise dendritic cells in BALF. With thisapproach, we identified CD1a+ dendritic cells as a subset ofmDCs in BALF. This CD1a+ subset of mDCs represented themajority of BALF mDCs, but was nearly absent amongperipheral blood mDCs, suggesting that CD1a is upregulatedduring the passage of mDCs into the airways. CD1a+ dendriticcells were previously described as intraepithelial dendritic cells,

Table 3 Expression of surface molecules on dendritic cell subsets

B1 (%) B2 (%) C1 (%) C2 (%) A2 (%)

mDCBDCA-1 62.8 (42.8 to 80.8) 63.5 (38.4 to 82.6) 81.3 (65.0 to 94.7) 85.9 (73.3 to 96.8) 92.6 (84.3 to 97.5)BDCA-2 0.0 (0.0 to 6.8) 0.2 (0.0 to 10.1) 0.0 (0.0 to 0.4) 0.0 (0.0 to 0.4) 0.8 (0.0 to 2.3)BDCA-3 19.6 (15.6 to 32.6) 23.8 (11.4 to 48.8) 73.1 (57.6 to 94.2) 76.1 (61.2 to 87.7) 87.5 (69.4 to 96.4)BDCA-4 0.0 (0.0 to 2.9) 0.0 (0.0 to 4.6) 20.0 (0.2 to 52.2) 23.3 (15.0 to 43.2) 45.1 (10.8 to 81.5)CD40 43.4 (13.4 to 54.2) 33.0 (11.2 to 75.8) 98.0 (93.1 to 100.0) 95.1 (92.9 to 99.3) 96.6 (90.1 to 99.1)CD80 0.0 (0.0 to 0.9) 0.0 (0.0 to 0.5) 70.8 (50.6 to 80.9) 58.6 (39.8 to 75.8) 43.3 (16.9 to 62.0)CD83 0.0 0.0 26.5 (19.9 to 33.3) 27.6 (12.7 to 38.1) 11.1 (7.3 to 25.1)CD86 1.0 (0.0 to 7.2) 1.5 (0.0 to 8.2) 84.3 (65.8 to 90.0) 87.0 (67.6 to 93.0) 78.9 (39.4 to 86.8)CD1a bd bd 71.8 (60.0 to 100.0) 69.6 (51.1 to 91.8) 72.9 (59.3 to 87.3)

pDCBDCA-1 0.0 0.0 NA NA 0.0 (0.0 to 3.4)BDCA-2 99.2 (94.7 to 99.7) 98.9 (96.1 to 100.0) NA NA 98.1 (91.1 to 100.0)BDCA-3 53.2 (17.3 to 73.5) 52.1 (28.4 to 65.5) NA NA 97.1 (94.9 to 100.0)BDCA-4 94.2 (92.3 to 99.7) 96.5 (92.9 to 100.0) NA NA 96.4 (77.4 to 99.5)CD40 88.4 (27.2 to 92.6) 82.6 (23.8 to 89.9) NA NA 57.1 (36.8 to 77.7)CD80 0.0 0.0 NA NA 9.5 (2.5 to 30.0)CD83 0.0 0.0 NA NA 0.0 (0.0 to 2.3)CD86 0.0 (0.0 to 0.6) 0.0 (0.0 to 3.2) NA NA 9.0 (0.6 to 12.5)

A2, bronchoalveolar lavage fluid (BALF) 24 h after allergen challenge; B1, peripheral blood before allergen challenge; B2, peripheral blood 24 h after allergenchallenge; bd, below detection limit; BDCA, blood dendritic cell antigen; C1, BALF 10 min after saline challenge; C2, BALF 24 h after saline challenge; mDC, myeloiddendritic cells; NA, not analysable; pDC, plasmacytoid dendritic cells.Values in parentheses denote ranges (minimum to maximum).

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with a yet undefined relationship with mDCs.2 Our data provideevidence that these cells represent a subgroup of mDCs inBALF.

Recently, the use of magnetic beads targeting blood dendriticcell antigens (BDCA 1–4) has been proposed as a straightfor-ward strategy to isolate subsets of dendritic cells from lunghomogenates10 11 and BALF.13 In peripheral blood, BDCA-2 andBDCA-4 are confined to pDCs, whereas BDCA-1 and BDCA-3are confined to mDCs.26 In agreement with this distribution inperipheral blood, BDCA-1 was confined to mDCs and BDCA-2to pDCs in BALF. However, there was a rather unspecificexpression of BDCA-3 and BDCA-4 in BALF. BDCA-3 was alsoexpressed on pDCs, BDCA-1+ mDCs and a variety of other BALFcells including a population of linbrightHLA-DR+ cells within thelymphocyte/dendritic cell gate. BDCA-4 was expressed onmDCs and a linbrightHLA-DR+ cell population in BALF. Thesedata suggest that BDCA-3 and BDCA-4 are not specific formDCs or pDCs in the BALF of patients with allergic asthma. Inaddition, the previously reported BDCA-3+HLA-DR+ dendriticcell subset in lung homogenates termed ‘‘mDC2’’10 11 might alsocontain pDCs. This hypothesis is supported by the findings thata subpopulation of mDC2 is CD11c2,10 and that the T cellproliferation induced by mDC2 ranges between that induced bymDC1 and pDC.11 Therefore, further analysis of the cellulardistribution of BDCA molecules in human BALF or lunghomogenates will be required before anti-BDCA-3 and anti-BDCA-4 beads can be recommended as a suitable tool to isolatesubsets of pulmonary dendritic cells.

There is accumulating evidence from animal models thatmDCs have a crucial role in the development and maintenanceof allergic asthma.3 6 In contrast, pDCs have been reported toinhibit allergic airway inflammation and Th2-type cytokineproduction in a mouse model of asthma.27 It has therefore beenhypothesised that mDCs and pDCs might be in a yin-yangbalance in allergic asthma, with a proallergic role of mDCs andantiallergic properties of pDCs. However, there is uncertaintywhether this concept is applicable to human asthma.Plasmacytoid dendritic cells are increased in the peripheralblood of patients with allergic asthma and other atopicdiseases, and are positively correlated with IgE levels andeosinophil counts.20 28 29 In patients with atopic rhinitis anddermatitis, there is a strong local increase in pDCs after allergenchallenge.18 19 Human pDCs can stimulate allergen-dependent Tcell proliferation and Th2-type cytokine production as effi-ciently as mDCs, but are also capable of Th1-type cytokineproduction after stimulation with CpG motifs.30 A recent reportsuggests that the decision as to whether pDCs will trigger eithera Th1-type or a Th2-type immune response may be dependenton the local microenvironment and pre-stimulation with Th1-type or Th2-type mediators.31 Therefore, the precise role of pDCsin human asthma is still unclear.

We show for the first time that allergen challenge isassociated with a marked influx of both mDCs and pDCs intoBALF in patients with asthma. Of note, the percentage increaseof pDCs in BALF was higher than that of mDCs in BALF, asreflected by an enhanced pDC:mDC ratio after allergenchallenge. In keeping with previous data,17 there was a decreasein mDCs in peripheral blood after allergen challenge. Inaddition, there was a trend to a decrease in peripheral bloodpDCs in this condition. These data suggest that both mDCs andpDCs are recruited from peripheral blood to the humanrespiratory tract after allergen challenge. Migration of imma-ture peripheral blood dendritic cells into the airways would alsoexplain the observation that the percentage of mature CD83+

mDCs was significantly reduced in BALF after allergenchallenge. However, the decrease in mature CD83+ mDCs inBALF might also be due to migration of mature endobronchial

mDCs to mediastinal lymph nodes after allergen challenge.Notably, the number of local effector cells (such as eosinophilsand lymphocytes) was correlated with the number of infiltrat-ing dendritic cells after allergen challenge. This finding suggeststhat the amount of dendritic cells recruited into the airwaysmight be linked to or triggered by airway inflammation. Thus,according to the concept that dendritic cells have a role inmaintaining an established airway inflammation in asthma,8

an inflammation-triggered recruitment of dendritic cells intothe airways could represent a vicious cycle in allergic asthma. Itremains to be elucidated, however, whether the recruited mDCsand pDCs have similar or opposing roles in this condition.

In conclusion, we described a strategy to comprehensivelyanalyse dendritic cell subsets and dendritic cell surfacemolecules in human bronchoalveolar lavage fluid. Using thisstrategy, we showed a marked local increase of not only mDCsbut also pDCs after allergen challenge in patients with asthma,which suggests that both dendritic cell subsets are involved inthe pathogenesis of asthma.

ACKNOWLEDGEMENTSWe thank Petra Thamm for excellent technical assistance.

Authors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Kai Bratke*, Marek Lommatzsch*, Peter Julius, Michael Kuepper, WernerLuttmann, J Christian Virchow, Department of Pneumology, University ofRostock, Rostock, GermanyHans-Dieter Kleine, Department of Hematology and Oncology, Universityof Rostock, Rostock, Germany

*Both authors contributed equally.

Funding: Deutsche Forschungsgemeinschaft (DFG) (Grant LO 1145/2-1).

Competing interests: None.

Ethical approval: This study was approved by the local ethics committee ofRostock, Germany.

REFERENCES1 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature

1998;392:245–52.2 Vermaelen K, Pauwels R. Pulmonary dendritic cells. Am J Respir Crit Care Med

2005;172:530–51.3 Lambrecht BN, Hammad H. Taking our breath away: dendritic cells in the

pathogenesis of asthma. Nat Rev Immunol 2003;3:994–1003.4 Lambrecht BN, Carro-Muino I, Vermaelen K, et al. Allergen-induced changes in

bone-marrow progenitor and airway dendritic cells in sensitized rats. Am J RespirCell Mol Biol 1999;20:1165–74.

5 Vermaelen KY, Cataldo D, Tournoy K, et al. Matrix metalloproteinase-9-mediated dendritic cell recruitment into the airways is a critical step in a mousemodel of asthma. J Immunol 2003;171:1016–22.

6 Lambrecht BN, De Veerman M, Coyle AJ, et al. Myeloid dendritic cells induceTh2 responses to inhaled antigen, leading to eosinophilic airway inflammation.J Clin Invest 2000;106:551–9.

7 van Rijt LS, Jung S, Kleinjan A, et al. In vivo depletion of lung CD11c+ dendriticcells during allergen challenge abrogates the characteristic features of asthma.J Exp Med 2005;201:981–91.

8 van Rijt LS, Lambrecht BN. Dendritic cells in asthma: a function beyondsensitization. Clin Exp Allergy 2005;35:1125–34.

9 Cochand L, Isler P, Songeon F, et al. Human lung dendritic cells have animmature phenotype with efficient mannose receptors. Am J Respir Cell Mol Biol1999;21:547–54.

10 Demedts IK, Brusselle GG, Vermaelen KY, et al. Identification andcharacterization of human pulmonary dendritic cells. Am J Respir Cell Mol Biol2005;32:177–84.

11 Demedts IK, Bracke KR, Maes T, et al. Different roles for human lung dendriticcell subsets in pulmonary immune defense mechanisms. Am J Respir Cell Mol Biol2006;35:387–93.

12 Donnenberg VS, Donnenberg AD. Identification, rare-event detection andanalysis of dendritic cell subsets in broncho-alveolar lavage fluid and peripheralblood by flow cytometry. Front Biosci 2003;8:s1175–80.

13 Tsoumakidou M, Tzanakis N, Papadaki HA, et al. Isolation of myeloid andplasmacytoid dendritic cells from human bronchoalveolar lavage fluid. ImmunolCell Biol 2006;84:267–73.

174 Bratke, Lommatzsch, Julius, et al

www.thoraxjnl.com

on Novem

ber 1, 2020 by guest. Protected by copyright.

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horax: first published as 10.1136/thx.2006.067793 on 23 August 2006. D

ownloaded from

14 Bellini A, Vittori E, Marini M, et al. Intraepithelial dendritic cells and selectiveactivation of Th2-like lymphocytes in patients with atopic asthma. Chest1993;103:997–1005.

15 Moller GM, Overbeek SE, Van Helden-Meeuwsen CG, et al. Increasednumbers of dendritic cells in the bronchial mucosa of atopic asthmatic patients:downregulation by inhaled corticosteroids. Clin Exp Allergy 1996;26:517–24.

16 Jahnsen FL, Moloney ED, Hogan T, et al. Rapid dendritic cell recruitment to thebronchial mucosa of patients with atopic asthma in response to local allergenchallenge. Thorax 2001;56:823–6.

17 Upham JW, Denburg JA, O’Byrne PM. Rapid response of circulating myeloiddendritic cells to inhaled allergen in asthmatic subjects. Clin Exp Allergy2002;32:818–23.

18 Jahnsen FL, Lund-Johansen F, Dunne JF, et al. Experimentally inducedrecruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy.J Immunol 2000;165:4062–8.

19 Bangert C, Friedl J, Stary G, et al. Immunopathologic features of allergic contactdermatitis in humans: participation of plasmacytoid dendritic cells in thepathogenesis of the disease? J Invest Dermatol 2003;121:1409–18.

20 Matsuda H, Suda T, Hashizume H, et al. Alteration of balance between myeloiddendritic cells and plasmacytoid dendritic cells in peripheral blood of patientswith asthma. Am J Respir Crit Care Med 2002;166:1050–4.

21 Lommatzsch M, Julius P, Kuepper M, et al. The course of allergen-inducedleukocyte infiltration in human and experimental asthma. J Allergy Clin Immunol2006;118:91–7.

22 Nassenstein C, Braun A, Erpenbeck VJ, et al. The neurotrophins nerve growthfactor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4are survival and activation factors for eosinophils in patients with allergicbronchial asthma. J Exp Med 2003;198:455–67.

23 van Haarst JM, de Wit HJ, Drexhage HA, et al. Distribution andimmunophenotype of mononuclear phagocytes and dendritic cells in the humanlung. Am J Respir Cell Mol Biol 1994;10:487–92.

24 van Haarst JM, Hoogsteden HC, de Wit HJ, et al. Dendritic cells and theirprecursors isolated from human bronchoalveolar lavage: immunocytologic andfunctional properties. Am J Respir Cell Mol Biol 1994;11:344–50.

25 van Haarst JM, Verhoeven GT, de Wit HJ, et al. CD1a+ and CD1a- accessorycells from human bronchoalveolar lavage differ in allostimulatory potential andcytokine production. Am J Respir Cell Mol Biol 1996;15:752–9.

26 Dzionek A, Fuchs A, Schmidt P, et al. BDCA-2, BDCA-3, and BDCA-4: threemarkers for distinct subsets of dendritic cells in human peripheral blood.J Immunol 2000;165:6037–46.

27 de Heer HJ, Hammad H, Soullie T, et al. Essential role of lung plasmacytoiddendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J ExpMed 2004;200:89–98.

28 Uchida Y, Kurasawa K, Nakajima H, et al. Increase of dendritic cells of type 2(DC2) by altered response to IL-4 in atopic patients. J Allergy Clin Immunol2001;108:1005–11.

29 Hashizume H, Horibe T, Yagi H, et al. Compartmental imbalance and aberrantimmune function of blood CD123+ (plasmacytoid) and CD11c+ (myeloid)dendritic cells in atopic dermatitis. J Immunol 2005;174:2396–403.

30 Farkas L, Kvale EO, Johansen FE, et al. Plasmacytoid dendritic cells activateallergen-specific TH2 memory cells: modulation by CpG oligodeoxynucleotides.J Allergy Clin Immunol 2004;114:436–43.

31 Schroeder JT, Bieneman AP, Xiao H, et al. TLR9- and FcepsilonRI-mediatedresponses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression. J Immunol 2005;175:5724–31.

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